User:Tk/Notebook/MF-xfm/2008/04/06: Difference between revisions

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(Autocreate 2008/04/06 Entry for User:Tk/Notebook/MF-xfm)
 
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==Entry title==
==Prepare electrocompetent M.florum cells==
* Insert content here...
 
* Grow 45 ml culture in 1161 medium, no antibiotic
** growth is to yellow, light turbid phase
* Chill culture on ice for 30 minutes while fast cooling centrifuge
* Spin down 10 min at 9000g
* remove supernatent, resuspend pellet
* Add 45 ml ice cold EPB
* leave on ice 15 minutes
* Spin down 10 minutes at 9000g
* remove supernatent, resuspend pellet
* Add 45 ml ice cold EPB
* leave on ice 15 minutes
* Spin down 10 minutes at 9000g
* remove all supernatent, resuspend pellet
* Bring pellet to 250 &mu;l volume (200x concentration)
* Add 25 &mu;l glycerol (10%)
* leave on ice for 30 minutes
* Aliquot 5x 50 &mu;l into 1.7 ml eppendorfs, prechilled on ice
* Flash freeze in dry ice/ethanol
* Store at -80C until use
 
==Make transposomes==
* Add 2 &mu;l 100 ng/&mu;l ME0 PCR DNA
* Add 4 &mu;l 500 ng/&mu;l transposase
* Add 2 &mu;l glycerol
* incubate at 37C for 1 hour
* hold at -20C indefinitely
* Made 80 &mu;l transposomes with ME0/PCR/PCR cleanup DNA 118 ng/&mu;l (20 &mu;l)
* Will test with E. coli transformations
* Will test with M. florum transformations
 
 
==Electroporation results==
* 1 ng/ul and 100 pg/ul E. coli transformations sparked at 2.5 KV
* 10 pg/ul and transposome E. coli transformations worked at 2.5 KV
* Me. florum transformations with transposomes sparked at 2.5 KV and 2.2 KV, functioned at 2.0 KV
* Outgrowth for  1 hour, plate on LB amp and LB Tet for E. coli, 1161 agar + tet for Me. florum




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Revision as of 21:42, 19 June 2008

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Prepare electrocompetent M.florum cells

  • Grow 45 ml culture in 1161 medium, no antibiotic
    • growth is to yellow, light turbid phase
  • Chill culture on ice for 30 minutes while fast cooling centrifuge
  • Spin down 10 min at 9000g
  • remove supernatent, resuspend pellet
  • Add 45 ml ice cold EPB
  • leave on ice 15 minutes
  • Spin down 10 minutes at 9000g
  • remove supernatent, resuspend pellet
  • Add 45 ml ice cold EPB
  • leave on ice 15 minutes
  • Spin down 10 minutes at 9000g
  • remove all supernatent, resuspend pellet
  • Bring pellet to 250 μl volume (200x concentration)
  • Add 25 μl glycerol (10%)
  • leave on ice for 30 minutes
  • Aliquot 5x 50 μl into 1.7 ml eppendorfs, prechilled on ice
  • Flash freeze in dry ice/ethanol
  • Store at -80C until use

Make transposomes

  • Add 2 μl 100 ng/μl ME0 PCR DNA
  • Add 4 μl 500 ng/μl transposase
  • Add 2 μl glycerol
  • incubate at 37C for 1 hour
  • hold at -20C indefinitely
  • Made 80 μl transposomes with ME0/PCR/PCR cleanup DNA 118 ng/μl (20 μl)
  • Will test with E. coli transformations
  • Will test with M. florum transformations


Electroporation results

  • 1 ng/ul and 100 pg/ul E. coli transformations sparked at 2.5 KV
  • 10 pg/ul and transposome E. coli transformations worked at 2.5 KV
  • Me. florum transformations with transposomes sparked at 2.5 KV and 2.2 KV, functioned at 2.0 KV
  • Outgrowth for 1 hour, plate on LB amp and LB Tet for E. coli, 1161 agar + tet for Me. florum


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